1. Field of the Invention
This invention relates specifically to a new assay procedure for the detection and measurement of certain biologically active substances, particularly immunochemical substances. This new assay procedure permits rapid qualitative and quantitative determinations to be made in an advantageous manner. The invention also relates to a novel separation technique that is of general utility, and more particularly, to kits useful for assay procedures.
2. Statement of the Prior Art and Other Information
There is a continuing need for rapid, accurate qualitative and quantitative determinations of many kinds of biologically active substances at extremely low concentrations, i.e., at physiological concentrations.
Today, there is for example a need for determining the presence of drugs or narcotics in body fluids, such as saliva, blood or urine. In addition, in medical diagnosis, it is frequently important to be able to detect and quantify the presence of various substances which are synthesized naturally by the body or ingested. These include hormones, both steroidal and polypeptides, prostaglandins, and toxins, as well as other materials which may be involved in body functions. Frequently, there is concern with extremely small amounts and occasionally, with very small differences in concentrations.
Beside these materials, assays effective at extremely low concentrations would be desirable for a variety of pesticides, such as insecticides, bactericides, fungicides, etc., as well as organic pollutants of other kinds, both in the air and water.
Various methods have been developed in the last two or three decades for the determination of a variety of immunochemical substances, including antigens, antibodies, haptens, and certain low molecular weight substances. Excellent surveys of the field are reported by: Skelley et al., Radioimmunoassay, Clinical Chemistry 19: 146-186 (1973); Wisdom, Enzyme-Immunoassay, Clinical Chemistry 22: 1243-1255 (1976); and Schuurs et al., Clin. Chem. Acta 81: 1-40 (1977). There is also a good description of assay techniques in U.S. Pat. No. 4,213,764, issued July 22, 1980.
Examples of some of the assay methods are:
1. Radioassay techniques PA0 2. Fluoroimmunoassays (FIA) PA0 3. Enzyme immunoassay (EIA) PA0 4. Latex-particle agglutination (LPA) PA0 5. Charcoal-particle agglutination (CPA) PA0 6. Hemagglutination and Hemagglutination Inhibition Assays (HA), (HIA) PA0 7. Radial Immunodiffusion and Double diffusion (RID) PA0 8. Viroimmunoassay (VIA); and PA0 9. Spin immunoassay (SIA), among others.
a. Competitive protein binding assays PA1 b. Radioimmunoassay (RIA) PA1 c. Immunoradiometric assays PA1 d. Sandwich or 2-site immunoradiometric assays
Many of the immunochemical assay systems involve the use of labels. There are many types of labels that are useful for the detection and measurement of biologically important or interesting compounds or substances in serum or other media.
The administration of most of these tests is hampered by one or more of the following limitations: (1) lack of sensitivity, (2) complexity of the test procedure, (3) instability of reagents, (4) hazardous nature of one or more reagents, (5) impure reagents, and (6) expensive equipment required to perform quantitative and qualitative analysis of the amount of label involved, as in an immunochemical reaction. For a review of the development and evaluation of immunological methods and their uses as diagnostic tools, reference is made to "Immunology as a Laboratory Tool" by Franz Peetoome American Journal of Technology 37: 445-469 (1971).
In addition to the general limitations mentioned above, it should be pointed out that the limitation as to "lack of sensitivity" is a very broad term. Some assay procedures have an acceptable sensitivity within one molecular weight range, but unacceptable sensitivity outside that range. Generally the previously available EIA assay techniques could be selected to provide acceptable sensitivity below 1,500 daltons (homo-geneous EIA) and above 60,000 daltons (heterogeneous EIA), but have not provided fully acceptable sensitivity for antigens, for example, having molecular weights between these two figures.
Both labeled and unlabeled immunochemical assay techniques may employ various devices to separate (1), immunochemical constituents which have reacted, from (2), nonreacted immunochemical constituents, and from (3), substances irrelevant to the test. An excellent survey of separation techniques, Separation Techniques in Saturation Analysis, by J. G. Ratcliff, appears at Br. Med. Bull. 30: 32-37 (1974).
For example, some patented EIA techniques require separation through the use of one component in the antigen-antibody reaction in an "insolubilized" phase for separation; see Schuurs and coworkers, in U.S. Pat. Nos. 3,654,090; 3,791,932; 3,850,752; 3,839,153; 3,879,262; 4,016,043 and Reissue 29,169; see also Ratcliff, supra, at pp. 35-36. The separation of bound and unbound antigen is a critical step in some radioimmunoassay (RIA) techniques as well as in some enzyme immunoassay (EIA) techniques. Marsden, Lab. Management, March: 31-34 (1977). Ratcliffe, supra. Odell et al., Proceedings of the Fifth Tenovus Workshop, Wales, U.K. pp. 207-222 (1975). Collins et al., Proceedings of the Fifth Tenovus Workshop, Wales, U.K. p. 223-225 (1975).
The most widely used separation method in RIA for small antigens is an adsorption technique. This system precipitates antibody unbound tracer using adsorbent materials, such as dextran coated charcoal, talc, or resins, and has advantages in its simplicity and reproducibility.
EIA techniques so far developed, however, rely mainly on the double antibody precipitation method. The double antibody method involves precipitation of antibody-bound enzyme-antigen conjugate using a second antibody produced against the immunoglobulin of the first animal. It is a most reliable and reproducible method. The double antibody method is described by Exley et al. in FEBS Letters 79 301-304 (1977) and FEBS Letters 91 162-165 (1978), and in Ratcliffe, supra, pp. 34-35. This method, however, often requires a long incubation time, frequent washings of the precipitates, and involves complex reaction kinetics. The double antibody technique was probably developed because it is so difficult to find suitable materials that will precipitate only unbound conjugate (i.e., enzyme-antigen, enzyme-hapten, or other enzyme-ligand conjugate); see Wisdom, supra, and Schuurs et al., supra.
Those assay techniques that require the presence of a solid phase, as for effecting separation, are commonly referred to as heterogeneous. Generally assays of this type are considered to have good sensitivity for antigens, other substances having high molecular weights, and like ligands. However such assays are not readily susceptible to automation, because of the need for centrifugation, or other separation step, to separate the solid and liquid phases, and because of the need for repeated washings. In a different assay technique referred to as the homogeneous technique, all of the materials remain in the liquid phase; no solid phase is used. This type of assay is generally considered to have good speed and good sensitivity, but not for antigens and other substances having high molecular weight. The homogeneous type of assay is generally limited as to the molecular weights of the ligands with which it is useful. Most types of homogeneous assay are considered to be useful with ligands of 1,500 daltons molecular weight (M.W.) or less. Moreover, homogeneous assays have been most successfully applied only above the nanogram/ml. level. They are easily adapted to automated clinical equipment and when so adapted, can have high sample processing capability (high throughput).
The homogeneous type of assay method does not require separation of free and bound label but rather depends on the inhibition or activation of the enzyme label by antibody binding (e.g., the EMIT R-type of Syva Corporation of Palo Alto, Calif., for EIA and FRAT, or "free radical assay technique", for SIA). Such assay techniques are described in U.S. Pat. Nos. 3,880,715; 3,852,157; 3,875,011; 3,935,074; and 3,905,871, and in an article by Kenneth S. Rubenstein et al., Homogeneous Enzyme Immunoassay, a New Immunochemical Technique, Biochemical and Biophysical Research Communications 47: 846-851 (1972). Other homogeneous assays having similar but not identical properties to EMIT and FRAT are also known. Unfortunately, homogeneous assays or other currently available similar assays, where the antibody modulates enzyme activity in the assay, suffer from the disadvantage that they are insensitive and unable to measure analytes of more than about 1,500 daltons.
The competitions assay, often used in RIA techniques, is now considered a classical and well known technique for detecting immunochemicals such as antigens at a very low concentrations. It is based upon the competition between labeled and unlabeled antigen for a fixed, limited amount of antibody. As applied to RIA, it is described by R. Yalow and S. Berson in J. Clin. Invest. 39: 1157 (1960). The amount of unlabeled antigen influences the distribution of the labeled antigen between antibody-bound (B) labeled antigen and antibody-free (F) labeled antigen. Generally, the greater the amount of unlabeled antigen that is present, the smaller the amount of labeled antigen that is able to combine with the antibody. In order to obtain conclusive results from the distribution, a good separation between B and F must be made. Methods used for this purpose include, for instance, chromatoelectrophoresis, as described by S. Berson and R. Yalow in The Hormones, edited by G. Pincus et al., Academic Press, New York (1964), vol IV, 557, or insolubilization of the antibodies. This insolubilization can be achieved by chemical means (crosslinking or covalent binding to an insoluble carrier) or by physical methods (adsorption to an insoluble carrier).
Of the limitations cited above, a most serious limitation, as reported in U.S. Pat. No. 4,213,764, has been lack of adequate sensitivity to detect some antigens. In general, three levels of sensitivity are recognizable. Low sensitivity techniques, where materials detected and measured exist in microgram/milliliter quantities, include RID, CPA, and LPA. Intermediate sensitivity techniques, where microgram/milliliter to nanogram/milliliter quantities of materials may be measured, include HIA, HA, FIA, SIA, VIA, and EIA. Until recently only RIA was able to measure with ultrasensitivity the picogram/milliliter to femtogram/milliliter region.
A great many of the techniques listed above require that some form of physically or chemically identifiable label be attached to one or more of the reagents in the assay system in order that the result of a test can be detected. RIA, FIA, EIA, VIA, and SIA all fall into this category. Radioactivity, fluorescent moieties, enzymes, complement, viruses, and electron spin labels are used respectively to generate some form of endpoint signal. The sensitivity with which these labels can be detected directly and fundamentally affects the useful ranges of the test systems using them.
The sensitivity with which a labeling moiety can be measured depends upon the nature of the signal that it generates, the ability to detect that signal, and the intensity of signal available per unit amount of marker molecule, i.e., its specific activity. The radioimmunoassay (RIA) method in its various forms has been recognized as a very sensitive system. The RIA method, unfortunately, has several serious, well recognized disadvantages. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 by L. E. M. Miles and C. N. Hales; see "Labelled Antibodies and Immunological Assay Systems", Lancet, II, 492 (1968), and Nature 219, 168 (1968).
Since then the EIA technique has been extensively investigated and developed. It is recognized as a potentially extremely sensitive technique, because of the inherent amplification potential of the enzyme label. That is, one molecule of enzyme can convert many molecules of its substrate, to generate the desired signal. Often the signal is a color development.
Among the patents that are representative of the state of the art in the detection and measurement of immunochemical substances by the use of an enzyme label are U.S. Pat. Nos. 3,654,090, 3,666,421, 3,791,932, 3,839,153, 3,850,752, 3,879,262, and 4,190,496.
Each patent and literature item cited in this application is incorporated herein by reference.